CN113461786A - Avian influenza virus-like particle vaccine, and preparation method and application thereof - Google Patents
Avian influenza virus-like particle vaccine, and preparation method and application thereof Download PDFInfo
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Abstract
The invention relates to an avian influenza virus-like particle vaccine which comprises an immunization amount of H5 subtype and/or H7 subtype avian influenza virus-like particle antigen and a pharmaceutically acceptable carrier, wherein the avian influenza virus-like particle antigen is formed by assembling HA, NA and M1 antigen proteins of H5 subtype and/or H7 subtype avian influenza virus. The avian influenza virus-like particle vaccine has good immunogenicity, and has better immune effect than a commercial inactivated vaccine with higher antigen content at low dose.
Description
Technical Field
The invention relates to the field of biological pharmacy, in particular to a virus-like particle vaccine for protecting against avian influenza virus, and a preparation method and application thereof.
Background
Virus-like particles (VLPs) are hollow particles with a size of 15-400 nm, which are assembled from structural proteins of viruses. VLPs can be prepared by expressing one (or more) structural protein(s) of a virus in vitro with high efficiency, allowing it to self-assemble into hollow particles that are morphologically similar to native viruses. The method mainly clones virus structural protein genes into expression vectors, and then transfers the vectors into prokaryotic or eukaryotic cells for expression.
Avian Influenza Virus (AIV) belongs to the family orthomyxoviridae, the genus Influenza, Influenza a Virus. Avian Influenza (AI) is a syndrome of infection and disease in birds caused by this virus. The international veterinary Office (OIE) and the national regulations on epidemic prevention of domestic animals and poultry classify the disease as class A virulent infectious disease. The disease is currently reported to occur in many countries and regions of the world, where Highly Pathogenic Avian Influenza (HPAI), particularly caused by strains of subtype H5, subtype H7, is more severe, is characterized primarily by sudden morbidity and high mortality, often resulting in total mortality in the feeding flock.
Up to now, vaccination with whole virus vaccines remains the most effective means for preventing and controlling the avian influenza virus epidemics. However, because the variation speed of avian influenza virus is far greater than the research and development and preparation speed of corresponding vaccines of variant strains, and cross immune protection is hardly generated between different subtypes, the production of avian influenza vaccines needs to be updated constantly, and the production period of corresponding vaccines of influenza virus epidemic strains is longer, the production cost is higher, so that the waste of manpower and material resources is caused, and an ideal prevention and control effect can not be achieved.
Although the avian influenza virus-like particle vaccine published in the literature in the prior art can generate better immune response, the avian influenza virus-like particle vaccine still cannot achieve the immune effect of the commercial whole virus vaccine, and the universal expression efficiency is lower, so that the preparation of the avian influenza virus-like particle vaccine with good immune effect, safety and controllable cost by screening an ideal avian influenza strain sequence is urgent, and the avian influenza virus-like particle vaccine also meets the requirements of effective prevention and control of major animal diseases and healthy and sustainable development of animal husbandry proposed by the nation.
Disclosure of Invention
In order to solve the defects of the prior art, the invention provides an avian influenza virus-like particle antigen, wherein the avian influenza virus-like particle antigen is assembled by HA, NA and M1 antigen proteins of H5 subtype avian influenza virus, and the HA, NA and M1 antigen proteins of the H5 subtype avian influenza virus are respectively coded by nucleotide sequences shown in Seq ID No.1, 2 and 3 or degenerate sequences thereof or nucleotide sequences shown in Seq ID No.4, 5 and 6 or degenerate sequences thereof.
The H5 subtype avian influenza virus-like particle antigen HAs good immunogenicity, can provide complete protection for H5 subtype avian influenza virus, and HAs the immune efficacy equivalent to or better than that of inactivated vaccine with the same HA antigen content.
The invention also provides an avian influenza virus-like particle vaccine composition, wherein the avian influenza virus-like particle vaccine composition comprises an immunizing amount of one or two avian influenza virus-like particle antigens of H5 subtype and a pharmaceutically acceptable carrier.
In one embodiment of the invention, in the avian influenza virus-like particle vaccine composition, the content of the antigen in the H5 subtype avian influenza virus-like particle is HA titer 6log 2-8 log 2.
The HA titer of the antigen content of the avian influenza virus-like particles of subtype H5 in the vaccine of the avian influenza virus-like particles of the present invention may be arbitrarily selected from 6.0log2, 6.1log2, 6.2log2, 6.3log2, 6.4log2, 6.5log2, 6.6log2, 6.7log2, 6.8log2, 6.9log2, 7.0log2, 7.1log2, 7.2log2, 7.3log2, 7.4log2, 7.5log2, 7.6log2, 7.7log2, 7.8log2, 7.9log2 and 8.0log 2.
The H5 subtype avian influenza virus-like particle antigen HAs good immunogenicity, can generate complete protection when the content is HA titer 6log2, can generate complete protection against avian influenza 14 days after immunization, and HAs better immune effect than inactivated vaccine with higher HA antigen titer.
As an embodiment of the present invention, in the avian influenza virus-like particle vaccine composition of the present invention, the pharmaceutically acceptable carrier includes an adjuvant selected from the group consisting of: (1) mineral oil, alumina gel adjuvant, saponin, alfvudine, DDA; (2) water-in-oil emulsion, oil-in-water emulsion, water-in-oil-in-water emulsion; or (3) a copolymer of a polymer of acrylic acid or methacrylic acid, maleic anhydride and an alkenyl derivative; and one or more of RIBI adjuvant system, Block co-polymer, SAF-M, monophosphoryl lipid A, Avridine lipid-amine adjuvant, Escherichia coli heat-labile enterotoxin, cholera toxin, IMS 1314, muramyl dipeptide, Montanide ISA 206, and Gel adjuvant; preferably, the saponin is Quil A, QS-21, GPI-0100; preferably, the adjuvant is a mineral oil adjuvant, which is used to prepare a water-in-oil emulsion. The adjuvant content is 5% -70% V/V, preferably from 30% -70% V/V, more preferably 66% V/V.
As one embodiment of the present invention, in the avian influenza virus-like particle vaccine composition of the present invention, the vaccine composition further comprises an immunizing amount of avian influenza virus-like particle antigen of subtype H7; the H7 subtype avian influenza virus-like particle antigen is assembled by H7 subtype avian influenza virus HA, NA and M1 antigen proteins, and the H7 subtype avian influenza virus HA, NA and M1 antigen proteins are respectively coded by nucleotide sequences shown in Seq ID No.7, 8 and 9 or degenerate sequences thereof.
The H7 subtype avian influenza virus-like particle antigen HAs good immunogenicity, can provide complete protection for H7 subtype avian influenza virus, and HAs the immune efficacy equivalent to or better than that of inactivated vaccine with the same HA antigen content.
In one embodiment of the invention, in the avian influenza virus-like particle vaccine composition, the content of the antigen in the H7 subtype avian influenza virus-like particle is HA titer 6log 2-8 log 2.
The HA titer of the antigen content of the avian influenza virus-like particles of subtype H7 in the vaccine of the avian influenza virus-like particles of the present invention may be arbitrarily selected from 6.0log2, 6.1log2, 6.2log2, 6.3log2, 6.4log2, 6.5log2, 6.6log2, 6.7log2, 6.8log2, 6.9log2, 7.0log2, 7.1log2, 7.2log2, 7.3log2, 7.4log2, 7.5log2, 7.6log2, 7.7log2, 7.8log2, 7.9log2 and 8.0log 2.
The H7 subtype avian influenza virus-like particle antigen HAs good immunogenicity, can generate complete protection when the content is HA titer 6log2, can generate complete protection against avian influenza 14 days after immunization, and HAs better immune effect than inactivated vaccine with higher HA antigen titer.
As one embodiment of the present invention, the pharmaceutically acceptable carrier includes drugs, immunostimulants, antioxidants, surfactants, colorants, volatile oils, buffers, dispersants, propellants, and preservatives; the immunostimulant includes alpha-interferon, beta-interferon, gamma-interferon, granulocyte macrophage colony stimulating factor (GM-CSF), macrophage colony stimulating factor (M-CSF), and interleukin 2(IL 2).
To prepare such compositions, methods well known in the art may be used.
The invention also relates to a method for preparing the avian influenza virus-like particle vaccine, wherein the method comprises the following steps:
cloning the HA, NA and M1 antigen protein genes of the avian influenza virus to the same vector;
transforming or transducing and recombining the vector obtained in the step (1) to obtain a recombinant baculovirus plasmid containing the HA, NA and M1 antigen protein genes;
step (3) transfecting the recombinant baculovirus plasmid containing the HA, NA and M1 antigen protein gene obtained in the step (2) into an insect cell sf9, and expressing the HA, NA and M1 antigen protein in series; and
and (4) separating the avian influenza virus-like particle antigen which is released to the supernatant of an extracellular culture medium after the self-assembly in insect cells is finished and assembled by the HA, NA and M1 antigen proteins, adding an adjuvant, and uniformly mixing to obtain the vaccine composition.
In one embodiment of the present invention, in the method for preparing an avian influenza virus-like particle vaccine according to the present invention, the avian influenza virus HA, NA, M1 antigen protein gene in the step (1) is an H5 subtype or H7 subtype avian influenza virus HA, NA, M1 antigen protein gene, the H5 subtype avian influenza virus HA, NA, M1 antigen protein gene is represented by sequence Seq ID nos. 1, 2, 3 or sequence Seq ID nos. 4, 5, 6, respectively, and the H7 subtype avian influenza virus HA, NA, M1 antigen protein gene is represented by sequence Seq ID nos. 7, 8, 9, respectively.
As an embodiment of the present invention, in the method for preparing a avian influenza virus-like particle vaccine according to the present invention, the vector in the step (1) is pFastBac I; the baculovirus plasmid in the step (2) is Bacmid.
The invention also relates to application of the avian influenza virus-like particle vaccine in preparation of a medicament for preventing and/or treating diseases caused by avian influenza viruses.
As an embodiment of the present invention, in the use of the present invention, the avian influenza virus is an H5 subtype and/or an H7 subtype avian influenza virus.
The administration object for preparing the medicine for preventing and/or treating the avian influenza virus infection comprises chicken.
The antigen of the avian influenza virus-like particle vaccine is a self-assembly body of surface antigen hemagglutinin HA, neuraminidase NA and matrix protein M1 of avian influenza virus. The invention utilizes an insect baculovirus expression system to produce the avian influenza virus-like particle antigen, has the advantages of high yield, low production cost, good immunogenicity, no biological safety risk and the like, can provide protective activity for H5 subtype 2.3.4.4d branch avian influenza and H5 subtype 2.3.2.1d branch avian influenza, and can also provide protective activity for H7 subtype avian influenza.
Detailed Description
Hereinafter, embodiments of the present invention will be described.
Definition of
"Antigen" refers to a substance that induces an immune response in the body, i.e., a substance that is specifically recognized and bound by an Antigen receptor (TCR/BCR) on the surface of T/B lymphocytes, activates T/B cells, proliferates and differentiates the T/B cells, produces an immune response product (sensitized lymphocytes or antibodies), and specifically binds to the corresponding product in vitro or in vivo.
"Virus-like particles (VLPs)" are particles assembled from one or more viral structural proteins and have similar external structure and antigenicity to viral particles, but do not contain viral genes.
The term "vaccine", "vaccine composition" as used herein refers to a pharmaceutical composition comprising an avian influenza virus-like particle antigen, which induces, stimulates or enhances the immune response of a chicken against avian influenza.
The term "immunizing amount" shall be understood as an "immunologically effective amount," also referred to as an immunoprotective amount or an amount effective to produce an immune response, of antigen effective to induce an immune response in a recipient, sufficient to prevent or ameliorate the signs or symptoms of disease, including adverse health effects or complications thereof. The immune response may be sufficient for diagnostic purposes or other testing, or may be suitable for use in preventing signs or symptoms of disease, including adverse health consequences or complications thereof caused by infection by a pathogen. Humoral immunity or cell-mediated immunity or both can be induced. The immune response of an animal to an immunogenic composition can be assessed indirectly, for example, by measuring antibody titers, lymphocyte proliferation assays, or directly by monitoring signs or symptoms after challenge with a wild-type strain, while the protective immunity provided by the vaccine can be assessed by measuring, for example, clinical signs such as mortality, reduction in morbidity, temperature values, overall physiological condition of the subject, and overall health and performance. The immune response may include, but is not limited to, induction of cellular and/or humoral immunity.
The term "pharmaceutically acceptable carrier" refers to all other ingredients in the vaccine composition of the present invention, except for the avian influenza virus antigen, that do not stimulate the body and do not hinder the biological activity and properties of the compound used, and preferably are adjuvants. The term "adjuvant" may include an alumina gel adjuvant; saponins (saponin), such as Quil A, QS-21(Cambridge Biotech Incorporation, Cambridge MA), GPI-0100(Galenica Pharmaceuticals Incorporation, Birmingham AL); a water-in-oil emulsion; an oil-in-water emulsion; a water-in-oil-in-water emulsion; polymers of acrylic acid or methacrylic acid; maleic anhydride and alkenyl (alkenyl) derivatives. The term "emulsion" may be based in particular on light liquid paraffin oil (European Pharmacopea type); isoprenoid oils (isoprenoid oils) resulting from the oligomerization of olefins, such as squalane (squalane) or squalene oil (squalene oil), in particular isobutene or decene; linear alkyl-containing esters of acids or alcohols, more particularly vegetable oils, ethyl oleate, propylene glycol di- (caprylate/caprate), glycerol tri- (caprylate/caprate) or propylene glycol dioleate; esters of branched fatty acids or alcohols, especially isostearic acid esters. The oil is used in combination with an emulsifier to form an emulsion. The emulsifiers are preferably nonionic surfactants, in particular esters of sorbitan, of mannide (such as, for example, anhydrous mannitol oleate), of aliphatic diols (glycols), of polyglycerols, of propylene glycol and of oleic acid, of isostearic acid, of ricinoleic acid or of hydroxystearic acid, which are optionally ethoxylated, and also polyoxypropylene-polyoxyethylene block copolymers, in particular the Pluronic products, in particular L121. See The description of The same and The reactive application of adjuvants by Hunter et al (Ed. by DES Stewart-Tull, John Wiley and Sons, New York,1995:51-94) and The description of Vaccine by Todd et al (1997,15: 564-570). For example, the SPT emulsion described on page 147 and the MF59 emulsion described on page 183 of Vaccine design, the Subunit and adivant propaach (Plenum Press,1995) written by Powell M and Newman M can be used. The term "polymer of acrylic or methacrylic acid" is preferably a crosslinked polymer of acrylic or methacrylic acid, in particular a polyalkenyl ether or polyalcohol crosslinked with a sugar (sugar), these compounds being known under the name Carbomer (Carbopol, trade name Carbopol) (Phameuropa,1996,8 (2)). Those skilled in the art can also see US2909462, which describes such acrylic polymers crosslinked with polyhydroxylated compounds having at least 3 hydroxyl groups, preferably not more than 8, wherein the hydrogen atoms of at least 3 hydroxyl groups are substituted by unsaturated aliphatic hydrocarbon groups (aliphatic radial) having at least 2 carbon atoms. Preferred groups are those containing 2 to 4 carbon atoms, such as vinyl, allyl and other ethylenically unsaturated groups (ethylenically unsaturated groups). The unsaturated groups may themselves contain other substituents, such as methyl. These products are sold under the name carbopol, (BF Goodrich, Ohio, USA) are particularly suitable. They are crosslinked with allyl sucrose or with allyl pentaerythritol. Among these, mention may be made of carbopols 974P, 934P and 971P, the most preferred being the use of carbopol 971P. The term "copolymers of maleic anhydride and alkenyl derivative" also contemplates the maleic anhydride and ethylene copolymers ema (monsanto), which are dissolved in water to give an acidic solution, neutralized, preferably to physiological pH, in order to give an adjuvant solution into which the immunogenic, immunogenic or vaccinal composition itself can be incorporated. The term "adjuvant" also includes, but is not limited to, the RIBI adjuvant system (Ribi Incorporation), Block co-polymer (CytRx, Atlanta GA), SAF-M (Chiron, Emeryville CA), monophosphoryl lipid A (monophosphoryl lipid A), Avridine lipoamine adjuvant, E.coli heat labile enterotoxin (recombinant or otherwise), cholera toxin, IMS 1314, muramyl dipeptide, Gel adjuvant, and the like. Preferably, the adjuvant comprises one or more of mineral oil, an alumina Gel adjuvant, a saponin, a water-in-oil emulsion, an oil-in-water emulsion, a water-in-oil-in-water emulsion, a polymer of acrylic acid or methacrylic acid, a copolymer of maleic anhydride and an alkenyl (alkenyl) derivative, a RIBI adjuvant system, a Block co-polymer, SAF-M, a monophosphoryl lipid A, Avridine lipid-amine adjuvant, escherichia coli heat labile enterotoxin, cholera toxin, IMS 1314, muramyl dipeptide, Montanide ISA 206, or Gel adjuvant.
"degenerate sequence": in molecular biology, the phenomenon that the same amino acid has two or more codons is called degeneracy of the codon (degeneracy), and such a sequence is called degenerate sequence.
"Gene recombination": refers to the recombination of genes that control different traits. Modern genetic engineering techniques carry out genetic recombination, also called recombinant DNA, in vitro by artificial design, with the aim of transferring a genetic gene in one individual cell to another individual cell DNA molecule of a different character, causing genetic variation. After the target gene from the donor is transferred into the recipient bacterium, the expression of the gene product can be carried out, thereby obtaining a product which is difficult to obtain by a common method.
"transformation" refers to the acquisition of a new genetic phenotype in a cell or a cultured recipient cell by the automated acquisition or artificial supply of exogenous DNA.
"transduction" means that when the virus is released from an infected (donor) cell and re-infects another (recipient) cell, the DNA transfer and gene recombination that occurs between the donor and recipient cells is transduction.
The term "preventing and/or treating" when referring to an avian influenza virus infection refers to inhibiting replication of avian influenza virus, inhibiting transmission of avian influenza virus, or preventing colonization of avian influenza virus in its host, as well as alleviating the symptoms of an avian influenza virus infected disease or disorder. Treatment is considered to be therapeutically effective if the viral load is reduced, the condition is reduced and/or the food intake and/or growth is increased.
The invention will be further described with reference to specific embodiments, and the advantages and features of the invention will become apparent as the description proceeds. These examples are illustrative only and do not limit the scope of the present invention in any way. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be made without departing from the spirit and scope of the invention.
The chemical reagents used in the examples of the present invention are all analytical reagents and purchased from the national pharmaceutical group. The experimental methods are conventional methods unless specified otherwise; the biomaterial is commercially available unless otherwise specified.
Example 1 expression of subtype H5 avian influenza Virus-like particles
1. Carrier engineering
NdeI, NotI, SalI and XbaI restriction sites are inserted into the 4413-4414 site of a commercial vector pFastBac I as a template. Adding 1 mul of D pn I enzyme into the PCR product to digest away the template plasmid, taking 5 mul to transform DH5 alpha according to a conventional method, and naming the successfully transformed plasmid as pFastBac mut.
2. Construction of three expression cassette Donor plasmids
An HA gene shown in a sequence table Seq ID No.1, an NA gene shown in a sequence table Seq ID No.2 and an M1 gene shown in a sequence table Seq ID No.3 are synthesized by Suzhou Jinzhi Biotechnology Limited, and enzyme cutting sites of incision enzymes BamHI and HindIII are added at the upstream and the downstream of the genes respectively. The synthesized HA gene, NA gene and M1 gene are cut by BamHI and HindIII enzyme and are connected with pFastBac mut cut by the same enzyme, the connection product is converted into DH5 alpha, and the correct plasmids are identified and named as pFastBac mut-HA-1, pFastBac mut-NA-1 and pFastBac mut-M1-1.
Respectively amplifying HA and NA expression cassettes by taking pFastBac mut-HA-1 and pFastBac mut-NA-1 as templates, inserting the HA expression cassettes into pFastBac mut-M1-1 plasmid, and naming the plasmid as pFastBac mut-M1-HA-1; then the NA expression cassette is inserted into pFastBac mut-M1-HA-1, and the plasmid is named as pFastBac mut-M1-HA-NA-1.
3. Construction and identification of recombinant Bacmid
Adding 2 mu l of pFastBac mut-M1-HA-NA-1 plasmid into DH10Bac competent cells, flicking and uniformly mixing, incubating on ice for 30min, thermally shocking at 42 ℃ for 45s, incubating on ice for 5min, adding 400 mu l of SOC culture medium at 37 ℃ and 200rpm for 4h, taking 100 mu l of bacterial liquid, coating the bacterial liquid on a plate containing IPTG/X-gal/kanamycin/tetracyclic/gentamicin three antibiotics, culturing at 37 ℃ for at least 48h, and picking white single bacterial colony to 5ml of kanamycin/tetracyclic/gentamicin three antibiotics liquid LB culture medium for shaking bacteria overnight when the blue-white bacterial colony is obvious. Taking 1 mul as a template for PCR identification of bacteria liquid the next day. The PCR product is correctly identified, and the reagent in the Tiangen plasmid miniprep kit is used for extracting the recombinant Bacmid, which is named Bacmid-M1-1.
4. Acquisition and passage of recombinant baculovirus
Recombinant Bacmid-M1-1 was transfected into insect cells sf 9. Reference to
Regent instructions for transfection, 72h after transfection, harvest cell supernatant marker rBac-M1-1P 1 after cytopathic.
Sf9 cells in logarithmic growth phase were grown at 0.9X 106And (3) inoculating the cell/dish with 10cm of cell culture dish, adding the recombinant baculovirus of P1 generation into the cell culture dish paved with sf9 according to the volume ratio of 1:20-1:40 after the cells are completely attached to the wall, continuously culturing at 27 ℃, harvesting the supernatant and marking as the recombinant baculovirus of P2 generation when the cytopathic effect is obvious about 72 hours, wrapping the recombinant baculovirus with tinfoil paper, and keeping the recombinant baculovirus in a refrigerator at 4 ℃ in a dark place for later use. The step is repeated to inoculate according to the proportion of 1:100-A group of baculovirus.
5. Expression and characterization of proteins
Inoculating Sf9 cells to the recombinant virus transferred to P4 according to the volume ratio of 1: 5-1: 10, inoculating for about 72-96h to obtain cells, and centrifuging to obtain a supernatant. The HA content of the extracellular supernatant was determined to be 12log 2. The results of transmission electron microscope observation show that the harvested protein presents a virus-like particle shape, is basically uniform in size and is in a hollow particle state.
In the same manner as above, HA gene shown in sequence table Seq ID No.4, NA gene shown in sequence table Seq ID No.5, and M1 gene shown in sequence table Seq ID No.6 were synthesized by Kingzhi Biotech, Inc. of Suzhou, and a recombinant baculovirus named rBac-M1-2 was constructed. Inoculating Sf9 cells to the recombinant virus transferred to P4 according to the volume ratio of 1: 5-1: 10, inoculating for about 72-96h to obtain cells, and centrifuging to obtain a supernatant. The HA content of the extracellular supernatant was determined to be 12log 2. The results of transmission electron microscope observation show that the harvested protein presents a virus-like particle shape, is basically uniform in size and is in a hollow particle state.
Example 2 preparation of subtype H5 avian influenza Virus-like particle vaccine
The virus-like particles harvested in example 1 were added to mineral oil adjuvant to prepare vaccine compositions, and the specific ratios are shown in table 1.
TABLE 1 proportioning of subtype H5 avian influenza virus-like particle vaccine composition
| Components | Vaccine 1 | Vaccine 2 | Vaccine 3 | Vaccine 4 | Vaccine 5 | Vaccine 6 |
| rBac-M1-1(HA content) | 6log2 | 8log2 | - | - | 6log2 | 8log2 |
| rBac-M1-2(HA content) | - | - | 6log2 | 8log2 | 6log2 | 8log2 |
| Adjuvant (V/V%) | 66％ | 66％ | 66％ | 66％ | 66％ | 66％ |
Example 3 immunogenicity test of avian influenza Virus-like particle vaccine subtype H5
80 SPF chickens of 21 days old are divided into 8 groups, each group comprises 10 SPF chickens, the 1 st group to the 6 th group are respectively injected with vaccines 1 to 6 prepared in immunization example 2 through neck subcutaneous injection, the 7 th group is injected with commercial inactivated vaccine (H5(Re-11+ Re-12) and H7(H7-Re2) trivalent inactivated vaccine subcutaneously, the content of H5HA is 8log2), the immunization dose is 0.3ml, and the 8 th group is injected with 0.3ml of physiological saline subcutaneously to be used as blank control. All test chickens were kept separately, blood was collected 14 days and 21 days after immunization, serum was separated, and HI antibody titer was determined. The detection results of different HI antibodies after immunization are shown in tables 2 and 3.
TABLE 2 immunogenicity test results 1 for subtype H5 avian influenza virus-like particle vaccine
Remarks are as follows: the standard antigen of H5(H5-Re-11) was used
The results show that the 14-day HI antibody titer mean values of the 1 st group, the 2 nd group, the 5 th group and the 6 th group of immunization groups are all higher than 6log2, so that the immune protection against the H5 subtype 2.3.4.4d branched infection can be effectively provided, and the immune effect is better than the synchronous immune effect of the 7 th group of commercial vaccine immunization groups; the average value of the 14-day HI antibody titer of the 7 th group of commercial vaccine immunization groups reaches more than 6log2, so that the vaccine can effectively provide immune protection against H5 subtype 2.3.4.4d branched infection; under the immunization of the groups 1 and 5 with HA dose lower than that of inactivated vaccine, the average value of HI antibody titer in 14 days can still exceed the synchronous immunization effect of the group 7 commercial vaccine immunization group; when the group 2, the group 6 and the group 7 commercial vaccines are immunized at the same dose, the group 2 and the group 6 immune responses are faster, and the average value of HI antibodies of the 7 commercial vaccine on 21 days can be reached on 14 days; under the same HA dose immunization, the HI antibody titer mean value of the group 5 and the group 6 multicomponent immunization groups is higher than that of the group 1 and the group 2 single component immunization groups. The single-component virus-like particle vaccines (vaccine 1 and vaccine 2) and the multi-component virus-like particle vaccines (vaccine 5 and vaccine 6) prepared in the embodiment 2 of the invention can provide ideal immune efficacy; compared with the commercial whole virus inactivated vaccine, the virus-like particle vaccine composition provided by the invention has better immune effect and quicker immune response; the multicomponent virus-like particle vaccines (vaccine 5 and vaccine 6) provided by the invention have better immune effect.
TABLE 3 immunogenicity test results 2 for subtype H5 avian influenza virus-like particle vaccine
Remarks are as follows: the standard antigen of H5(H5-Re-12) was used
The results show that the 14-day HI antibody titer mean values of the 3 rd, 4 th, 5 th and 6 th immunization groups are all higher than 6log2, so that the vaccine can effectively provide immune protection against H5 subtype 2.3.2.1d branched infection, and the immune protection exceeds the synchronous immune effect of the 7 th commercial vaccine immunization group; the average value of the 14-day HI antibody titer of the 7 th group of commercial vaccine immunization groups reaches more than 6log2, so that the vaccine can effectively provide immune protection against H5 subtype 2.3.2.1d branched infection; under the immunization of the groups 3 and 5 with HA dose lower than that of inactivated vaccine, the average value of HI antibody titer in 14 days can still exceed the synchronous immunization effect of the group 7 commercial vaccine immunization group; when the 4 th group, the 6 th group and the 7 th group of commercial vaccines are immunized at the same dose, the 2 nd group and the 6 th group have faster immune response, and the average value of HI antibodies of the 7 th group of commercial vaccines at 21 days can be reached at 14 days; the HI antibody titers of the group 5 and 6 multicomponent immunization groups were higher than those of the group 3 and 4 single component immunization groups at the same HA dose immunization. The virus-like particle vaccines (vaccine 3 and vaccine 4) prepared in the embodiment 2 can provide ideal immune efficacy; compared with the commercial whole virus inactivated vaccine, the virus-like particle vaccine composition provided by the invention has better immune effect and quicker immune response; the multicomponent virus-like particle vaccines (vaccine 5 and vaccine 6) provided by the invention have better immune effect.
The virus-like particle vaccines provided by the invention have a good immune effect, and the multi-component virus-like particle vaccines generate a synergistic effect, so that the immune effect is better.
Example 4 expression of subtype H7 avian influenza Virus-like particles
An HA gene shown in sequence Listing Seq ID No.7, an NA gene shown in sequence ID No.8, and an M1 gene shown in sequence ID No.9 were synthesized by Kinzhi Biotech, Suzhou, and a recombinant baculovirus named rBac-M1-3 was constructed by the method of example 1. Inoculating Sf9 cells to the recombinant virus transferred to P4 according to the volume ratio of 1: 5-1: 10, inoculating for about 72-96h to obtain cells, and centrifuging to obtain a supernatant. The HA content of the extracellular supernatant was determined to be 12log 2. The results of transmission electron microscope observation show that the harvested protein presents a virus-like particle shape, is basically uniform in size and is in a hollow particle state.
Example 5 preparation of subtype H7 avian influenza Virus-like particle vaccine
The virus-like particles harvested in example 1 and example 4 were added to mineral oil adjuvant to prepare vaccine compositions, and the specific ratios are shown in table 1.
TABLE 4 proportioning of the subtype H7 avian influenza virus-like particle vaccine composition
Example 6 immunogenicity test of avian influenza Virus-like particle vaccine subtype H7
60 SPF chickens of 21 days old are divided into 6 groups, each group comprises 10 SPF chickens, the 9 th group to the 12 th group are respectively injected with vaccines 7 to 10 prepared in immunization example 5 through neck subcutaneous injection, the 13 th group is injected with commercial inactivated vaccine (H5(Re-11+ Re-12) and H7(H7-Re2) trivalent inactivated vaccine subcutaneously, the H7HA content is 8log2), the immunization dose is 0.3ml, and the 14 th group is injected with 0.3ml of physiological saline subcutaneously as blank control. All test chickens were kept separately, blood was collected 14 days and 21 days after immunization, serum was separated, and HI antibody titer was determined. The results of the different HI antibody detection after immunization are shown in tables 5, 6 and 7.
TABLE 5 immunogenicity test results 1 for subtype H7 avian influenza Virus-like particle vaccine
Remarks are as follows: the standard antigen of H7(H7-Re2) was used
The results show that the 14-day HI antibody titer mean values of the 9 th, 10 th, 11 th and 12 th immunization groups are all higher than 6log2, so that the vaccine can effectively provide immune protection against H7 subtype infection, and the synchronous immune effect is better than that of the 13 th group of commercial vaccine immunization groups; the average value of the HI antibody titer of the group 13 commercial vaccine immunization group at 14 days is more than 6log2, thus effectively providing immune protection against H7 subtype infection; under the immunization of the groups 9 and 11 with HA dose lower than that of inactivated vaccine, the average value of HI antibody titer in 14 days can still exceed the synchronous immunization effect of the group 7 commercial vaccine immunization group; when the groups 10, 12 and 13 commercial vaccines are immunized at the same dose, the 10 and 12 immune responses are faster, and the average HI antibody value of the 13 commercial vaccine on 21 days can be reached on 14 days. It is shown that the virus-like particle vaccines (vaccine 7, vaccine 8) prepared in example 5 of the present invention, whether single-component or multi-component, provide ideal immune efficacy; compared with the commercial whole virus inactivated vaccine, the virus-like particle vaccine composition provided by the invention has better immune effect and quicker immune response.
TABLE 6 immunogenicity test results 2 for subtype H7 avian influenza Virus-like particle vaccine
Remarks are as follows: the standard antigen of H5(H5-Re-11) was used
The results show that the 14-day HI antibody titer mean values of the 11 th group and the 12 th group of immunization groups are higher than 6log2, so that the immune protection against the H5 subtype 2.3.4.4d branched infection can be effectively provided, and the synchronous immune effect of the group 13 of commercial vaccine immunization groups is exceeded; the average value of the HI antibody titer of the group 13 commercial vaccine immunization group at 14 days is more than 6log2, thus effectively providing immune protection against H5 subtype 2.3.4.4d branched infection; under the condition of HA dose immunization lower than that of inactivated vaccine in the 11 th group, the average value of HI antibody titer in 14 days can still exceed the synchronous immunization effect of the commercial vaccine immunization group in the 13 th group; when the group 12 immunization group and the group 13 commercial vaccine group are immunized at the same dose, the group 12 immune response is generated more quickly, and the average value of HI antibodies of the group 13 commercial vaccine on 21 days can be reached on 14 days. The multicomponent virus-like particle vaccines (vaccine 9 and vaccine 10) prepared in the example 5 of the invention can provide ideal immune efficacy; compared with the commercial whole virus inactivated vaccine, the virus-like particle vaccine composition provided by the invention has better immune effect and quicker immune response.
TABLE 7 immunogenicity test results of H7 subtype avian influenza Virus-like particle vaccine 3
Remarks are as follows: the standard antigen of H5(H5-Re-12) was used
The results show that the 14-day HI antibody titer mean values of the 11 th group and the 12 th group of immunization groups are higher than 6log2, so that the immune protection against the H5 subtype 2.3.2.1d branched infection can be effectively provided, and the synchronous immune effect of the group 13 of commercial vaccine immunization groups is exceeded; the average value of the HI antibody titer of the group 13 commercial vaccine immunization group at 14 days is more than 6log2, thus effectively providing immune protection against H5 subtype 2.3.2.1d branched infection; under the condition of HA dose immunization lower than that of inactivated vaccine in the 11 th group, the average value of HI antibody titer in 14 days can still exceed the synchronous immunization effect of the commercial vaccine immunization group in the 13 th group; when the group 12 immunization group and the group 13 commercial vaccine group are immunized at the same dose, the group 12 immune response is generated more quickly, and the average value of HI antibodies of the group 13 commercial vaccine on 21 days can be reached on 14 days. The multicomponent virus-like particle vaccines (vaccine 9 and vaccine 10) prepared in the example 5 of the invention can provide ideal immune efficacy; compared with the commercial whole virus inactivated vaccine, the virus-like particle vaccine composition provided by the invention has better immune effect and quicker immune response.
In conclusion, the virus-like particle vaccine provided by the invention can provide ideal immune efficacy no matter a single-component virus-like particle vaccine or a multi-component virus-like particle vaccine, and compared with a commercial whole virus inactivated vaccine, the virus-like particle vaccine composition provided by the invention has better immune effect and quicker immune response. Example 7 duration of avian influenza Virus-like particle vaccine immunization test
30 SPF chickens of 21 days old are divided into 3 groups, 10 groups, 15 groups are injected with vaccine 9 prepared in immunization example 5 subcutaneously in the neck, 16 groups are injected with commercial inactivated vaccine (H5(Re-11+ Re-12) and H7(H7-Re2) trivalent inactivated vaccine subcutaneously, the contents of H5 and H7HA are both 8log2), the immunization dose is 0.3ml, and 17 groups are injected with 0.3ml of physiological saline subcutaneously as blank control. All test chickens were kept separately, blood was collected 21 days, 56 days, 84 days, 112 days, 140 days, 168 days, 196 days, and 224 days after immunization, serum was separated, and HI antibody titer was measured. The results of the different HI antibody detection after immunization are shown in tables 8, 9 and 10.
TABLE 8 avian influenza Virus-like particle vaccine immunization duration test results 1
Remarks are as follows: the standard antigen of H5(H5-Re-11) was used
The results show that the vaccine 9 immunization group has longer immunization duration than the commercial vaccine immunization group, and the HI antibody titer mean value is still higher than 6log2 at 32 weeks (224 days), so that the complete protection against the H5 subtype 2.3.4.4d branch can be achieved. The high-efficiency expression virus-like particle vaccine provided by the invention is shown to have good immunogenicity, and maintain long-term immunity.
TABLE 9 avian influenza Virus-like particle vaccine immunization duration test results 2
Remarks are as follows: the standard antigen of H5(H5-Re-12) was used
The results show that the vaccine 9 immunization group has longer immunization duration than the commercial vaccine immunization group, and the HI antibody titer mean value is still higher than 6log2 at 32 weeks (224 days), so that the complete protection against the H5 subtype 2.3.2.1d branch can be achieved. The high-efficiency expression virus-like particle vaccine provided by the invention is shown to have good immunogenicity, and maintain long-term immunity.
TABLE 10 avian influenza Virus-like particle vaccine immunization duration test results 3
Remarks are as follows: the standard antigen of H7(H7-Re2) was used
The results show that the vaccine 9 immunization group has longer immunization duration than the commercial vaccine immunization group, and the HI antibody titer mean value is still higher than 6log2 at 32 weeks (224 days), so that complete protection against the H7 subtype can be achieved. The high-efficiency expression virus-like particle vaccine provided by the invention is shown to have good immunogenicity, and maintain long-term immunity.
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims.
SEQUENCE LISTING
<110> Puleco bioengineering GmbH
<120> avian influenza virus-like particle vaccine, and preparation method and application thereof
<160> 9
<170> PatentIn version 3.3
<210> 1
<211> 1692
<212> DNA
<213> subtype H5 avian influenza virus
<400> 1
atggaaaaga tcgtgctgct gctcagcgtg gtgtccctgg ttaagtccga ccagatctgc 60
atcggatacc acgccaacaa ctccactgaa caagtggaca ccatcatgga gaagaacgtt 120
accgttactc acgctcagga catcctggag aagacccaca acggcaagct gtgtgacctg 180
aacggtgtga agcccctgat cctgaaggac tgtagcgtgg ctggatggct gctcggtaac 240
ccaatgtgtg acgagttcat ccgtgtgcca gagtggtcct acatcgtcga acgcgctaac 300
ccttccaacg acctgtgcta ccctggcaac ctgaacgact acgaagaact gaagcacctg 360
ctctctcgca ttaaccactt cgaaaaaacc cagatcatcc ccaagagatc ctggtctaac 420
cacacttcat ccggtgtgag cgctgcctgc ccataccagg gtgtggcctc cttcttccgc 480
aacgttgttt ggctgaccaa gaaaaacgac gcctacccta caatcaagat gtcatacaac 540
aacactaaca aggaggacct cctgatcttg tggggaatcc accactccaa ctccgctgaa 600
gaacagacta acctgtacaa gaaccctacc acctacgtga gcgtgggtac ctctaccctg 660
aaccaacgtc tggtgcctaa gatcgccact cgtagccaag tcaacggaca gaggggccgt 720
atggacttct tctggacaat cctccgccct aacgacgcca tccacttcga gtccaacggt 780
aacttcatcg ctcctgaata cgcctacaag atcatcaaga caggagacag cactatcatg 840
aagagcgaaa tcgaatacgg taactgtaac actaagtgcc agactcccat cggcgctatc 900
aactcatcca tgcctttcca caacatccac ccactcacta tcggtgaatg ccctaagtac 960
gtgaagagca acaagctggt gctggccaca ggcctgcgta actcccctct gcgtgaaaag 1020
aggggcctgt tcggtgctat cgctggcttc atcgagggcg gctggcaggg aatggttgac 1080
ggctggtacg gctaccacca ctctaacgag cagggtagcg gttacgctgc tgaccgtgag 1140
tccacacaga aggctatcga cggtgtgaca aacaaggtga actccatcat cgataagatg 1200
aacacccagt tcgaagccgt gggccgtgag ttcaactccc tcgaacgtcg catcgagaac 1260
ctcaacaaga agatggaaga cggtttcctc gacgtgtgga cctacaacgc cgaactgttg 1320
gtgctcatgg aaaacgagcg cactctggac ttccacgaca gcaacgtgaa gaacctgtac 1380
gacaaggtgc gtttgcagct ccgcgacaac gctaaggagt tgggaaacgg ttgtttcgag 1440
ttctaccaca agtgtgacaa cgaatgtatg gagtcagtgc gcaacggtac atacgactac 1500
cctcaatact ctgaagaggc ccgtctcaag cgtgaggaaa tctctggagt taagctggag 1560
tctatcggta cctaccagat cctgtccatc tactccacag ttgcttcctc cttggtcttg 1620
gctatcatcg tcgccggcct cagcctctgg atgtgttcca acggttccct ccagtgtcgc 1680
atctgcatct aa 1692
<210> 2
<211> 1380
<212> DNA
<213> subtype H5 avian influenza virus
<400> 2
atgaacccta accagaagat cacctgtatc agcgctacag gtgttacatt gagcgttgta 60
agcctgctga tcggtatcgc caacctgggt ctgaacatcg gactgcacta caaggtttct 120
gattctacca caatcaacat ccctaagact aacgaaacta acccaacaac cactaacatc 180
acaaacatca tcgttaacaa gaacgaggaa aagacattcc tgaagttgac taagcctctg 240
tgtgaagtga actcatggca catcttgtct aaggacaacg ccatccgtat cggcgaagac 300
gctcacatcc tcgttacacg cgaaccatac ctgagctgtg acccccaggg ttgccgtatg 360
ttcgccctga gccaaggcac caccctccgt ggccagcacg ctaacggtac tatccacgac 420
cgtagcccat tccgcgctct catcagttgg gaaatgggtc aggctccttc cccttacaac 480
acccgtgtgg aatgcatcgg ctggtcctcc acaagctgtc acgacggcat cagccgcatg 540
agcatctgta tctcaggtcc gaacaacaac gcttccgccg tggtctggta ccgtggtcgt 600
cctgtgaccg agatccctag ctgggccggc aacatcctgc gtacacacga gtccgagtgc 660
gtgtgccata agggcatctg ccccgtggtt atgactgacg gccccgccaa caacaaggcc 720
gctaccaaga tcatctactt caaggagggc atgatccaga aggctgaaga actgcagggt 780
aacgcccagc acatcgaaga atgctcctgt tacggtgctg cccgcatgat caagtgtgtg 840
tgccgtgaca actggaaggg cgctaaccgc cccgtgatca tcatcgaccc agagatgatg 900
actcacactt ccaagtacct gtgttccaag atcctgaccg acacttcccg ccctaacgac 960
ccaaccaacg gcaactgcga cgcccccatc accggtggca gcccagaccc aggtgtgaag 1020
ggcttcgctt tcctggacgg tgagaactcc tggctgggcc gtaccatctc taaggacagc 1080
cgttccggct acgagatgct gaaggtgcct aacgctgaga tcgacactca gtccggtcct 1140
atcagctacc agctgatcgt gaacaaccag aactggagcg gttactccgg cgccttcatc 1200
gactactggg ccaacaagga gtgcttcaac ccctgcttct acgttgaact gatccgtggt 1260
cgccctaagg aatccggcgt gctgtggacc agcaacagca tggtggccct gtgtggtagc 1320
cgtgagcgtc tcggttcctg gagctggcac gacggcgctg agatcatcta cttcaagtaa 1380
<210> 3
<211> 759
<212> DNA
<213> subtype H5 avian influenza virus
<400> 3
atgagcctgc tgaccgaagt tgaaacttac gttctgtcaa tcatccctag cggtcctctg 60
aaggctgaaa tcgctcagaa gctcgaagac gttttcgccg gaaagaacgc tgatctggag 120
gctttgatgg aatggctgaa gacccgccct atcctgtccc ctctgactaa gggcatcctg 180
ggtttcgtgt tcaccctgac cgtgcctagc gaacgcggcc tgcagcgtcg ccgcttcgtg 240
cagaacgccc tgaacggtaa cggtgaccct aacaacatgg accgcgctgt gaagctgtat 300
aagaagctga agcgtgagat caccttccac ggtgctaagg aggtggccct gagctacagc 360
accggtgctc tggcttcctg tatgggtctg atctacaacc gtatgggcac cgtgaccact 420
gaggtggctt tcggcctggt gtgtgccact tgtgaacaga tcgctgactc ccagcaccgt 480
agccaccgcc agatggctac catcaccaac cctctgatcc gtcacgaaaa ccgtatggtg 540
ctggcctcca ccaccgctaa ggccatggaa cagatggccg gttcctccga acaggctgct 600
gaagctatgg aggtcgctaa ccaagctagg cagatggtgc aggctatgag gactatcgga 660
actcacccta actcatccac aggactccgt gataaccttc tcgaaaacct ccaggcttac 720
caaaagcgta tgggtgttca aatgcaacgt ttcaagtaa 759
<210> 4
<211> 1695
<212> DNA
<213> subtype H5 avian influenza virus
<400> 4
atggagaaga tcgtcctgtt cttcgctaca atcagcctgg ttaagtccga ccacatctgt 60
atcggttacc acgctaacaa ctctacagag caggtggaca ccatcatgga gaagaacgtt 120
acagtcaccc acgctaagga catcctggaa aagactcaca acggcaagct gtgtgacctg 180
aacggtgtga agcccctcat cctgaaggac tgcagcgtgg ctggttggct gctcggtaac 240
ccactgtgtg acgagttcac caacgttccc gagtggtcct acatcgttga gaaggctaac 300
cctgctaacg acctgtgcta ccccggaaac ttcaacgact acgaagagct gaagcacctg 360
ctgtcccgca tcaaccactt cgaaaagatc cagatcatcc ccaaggacag ctggagcgac 420
cacgaagcct ccctcggcgt gtctgctgct tgtagctacc agggcaactc ctcattcttc 480
cgcaacgtgg tgtggctgat caagaaggac aacgcttacc ctacaatcaa gaagtcctac 540
aacaacacta acagggaaga tttgctgatc ctgtggggca tccaccaccc taacgacgaa 600
gccgagcaga caaagctcta ccagaaccct acaacttaca tctcaatcgg tacatccact 660
ctgaaccagc gtctcgtgcc aaagatcgcc actcgttcta agatcaacgg ccagtcagga 720
cgcatcgact tcttctggac aatcctcaag cccaacgacg ccatccactt cgaatcaaac 780
ggcaacttca tcgcccctga atacgcttac aagatcgtta agaagggtga ctccactatc 840
atgcgttcag aggttgaata cggtaactgc aacactcgct gccaaacccc tatcggcgcc 900
atcaacagct ctatgccttt ccacaacatc cacccattga caatcggaga atgtcctaag 960
tacgttaagt ctaacaagtt ggttctggcc acaggtctgc gcaacagccc cctccgcgaa 1020
aagcgcggcc tgttcggagc catcgctggt ttcatcgagg gtggctggca gggtatggtt 1080
gacggctggt acggttacca ccactccaac gagcaaggct ccggatacgc cgctgataag 1140
gagagcaccc agaaggccat cgatggagtg actaacaagg tgaactccat catcgacaag 1200
atgaacaccc agttcgaagc cgtgggccgt gagttcaaca acctggaacg tcgcatcgag 1260
aacctgaaca agaagatgga agacggtttc ctggacgtct ggacttacaa cgctgaactc 1320
ctggtcctga tggaaaacga acgcaccctg gacttccacg actccaacgt gaagaacctg 1380
tacgacaagg tccgtctcca actgaaggac aacgctaagg aactgggaaa cggttgcttc 1440
gagttctacc acaagtgtaa caacgaatgt atggagagcg tgaagaacgg tacctacgac 1500
taccctcagt acagcgaaga agctcgcctg aagcgtgaag aaatctccgg cgtgaagctg 1560
gagtctatcg gcatctacca gatcctgtct atctacagca cagtggcttc cagcctcgtg 1620
ctggccatca tgatggccgg tctgagcctc tggatgtgta gcaacggttc cctgcagtgt 1680
cgcatctgta tctaa 1695
<210> 5
<211> 1350
<212> DNA
<213> subtype H5 avian influenza virus
<400> 5
atgaacccta accagaagat cgtgaccatc ggtagcatct gcatggtcat cggtatcatc 60
agcctgatgc tgcagatcgg caacatcatc tccatctggg tgagccactc tatccaaact 120
ggcaaccagc accaaaccga accaatccgt aacactaact tcctgactga gaacacagtc 180
gcttctgtta cactggccgg aaactcttct ttgtgcccca tcaagggctg ggctgtgcac 240
tccaaggaca acagcatccg catcggttcc aagggcgacg tgttcgtgat ccgtgagccc 300
ttcatctctt gtagccacct ggagtgtcgc acattcttcc tgacccaggg tgccctcctg 360
aacgacaagc actccaacgg tactgttaag gaccgcagcc cacaccgcac actcatgagc 420
tgccctatcg gcgaggctcc cagcccatac aacagcaagt tcgaatcggt ggcctggtcc 480
gccagcgctt gtcacgacgg cacctcatgg ctcgtgatcg gtatcagcgg ccctgacaac 540
ggtgccgtgg ctgtgctgaa gtacaacggt atcatcaccg acactatcaa gtcttggcgt 600
aacaacatcc tgcgcaccca ggaaagcgag tgtgcctgcg tgaacggctc ctgcttcact 660
gtgatgactg acggtccttc caacggccag gcctcctaca agatcttcaa gatcgaaaag 720
ggcaaggtgg tgaagtccgt cgagctgaac gctcctaact accactacga ggagtgcagc 780
tgctaccctg aagccggcga agtgatgtgc gtgtgccgcg acaactggca cggtagcaac 840
cgcccttggg tgagcttcaa ccagaacctg gagtaccaga tcggttacat ctgtagcggt 900
gtgttcggcg acaacccccg tcctaacgac ggcacaggtt cctgtggtcc tatgagcagc 960
aacggcgctt acggcgtcaa gggcttctcc ttcaagtacg gtaacggtgt gtggatcgga 1020
cgtactaagt ccacccacag ccgtagcgga ttcgaaatga tctgggaccc taacggctgg 1080
accggaaccg actccgagtt cagcatgaag caggacatcg tcgctatcac agactggagc 1140
ggttacagcg gttccttcgt gcagcaccca gagctgaccg gtctggactg tatccgccct 1200
tgtttctggg ttgaactgat ccgtggccaa cctaaggagt ccacaatctg gactagcggt 1260
agtagcatta gcttctgtgg cgttaatagt gacaccgtta gctggtcctg gcctgacggc 1320
gctgaactgc cctttaccat tgataagtaa 1350
<210> 6
<211> 759
<212> DNA
<213> subtype H5 avian influenza virus
<400> 6
atgtctctgc tgactgaggt tgaaacctac gttttatcta ttatccccag cggaccactc 60
aaggctgaaa tcgctcagaa gctcgaagac gtcttcgctg gcaagaacac cgacctcgaa 120
gccctgatgg agtggctgaa gacccgtcca atcctgagcc cactgactaa gggcatcctg 180
ggattcgtct tcactctgac cgtccctagc gagcgcggcc tgcaacgccg ccgcttcgtg 240
cagaacgctc tgaacggtaa cggagaccca aacaacatgg accgcgccgt gaaactgtac 300
aaaaagctca agcgcgaaat cacattccac ggagccaagg aggtggccct gtcctattct 360
actggcgcct tggccagctg catgggactc atttacaatc gtatgggcac cgttacaact 420
gaagtggctt tcggtctggt gtgcgctaca tgtgagcaaa tcgccgactc acagcaccgt 480
tcccaccgcc agatggccac catcaccaac cctctgatcc gtcacgaaaa ccgcatggtg 540
ctggccagca ccaccgctaa ggccatggaa cagatggctg gtagctccga gcaggccgct 600
gaagctatgg aagtggctaa ccaggcccgt cagatggtgc aggctatgcg taccatcggc 660
acccacccta actcctccgc tggtctgcgt gacaacctgc tggaaaacct gcaggcttac 720
cagaagcgta tgggtgtgca gatgcagcgt ttcaagtaa 759
<210> 7
<211> 1683
<212> DNA
<213> subtype H7 avian influenza virus
<400> 7
atgaacaccc agatcctggt gttcgccctg atcgccatca tccctactaa cgctgacaag 60
atctgtctcg gccaccacgc tgtgagcaac ggtaccaagg tgaacaccct gaccgagaag 120
ggtgtggaag ttgtgaacgc caccgagacc gtggaacgca ctaacacccc ccgcatctgc 180
tctaagggca agcgcaccgt ggacctgggc cagtgcggct tgctgggcac tatcaccggt 240
ccccctcagt gcgaccagtt cctggagttc agcgctgacc tgatcatcga gcgccgtgag 300
ggcagcgacg tgtgttaccc tggcaagttc gtgaacgaag aggctctccg tcagatcctg 360
cgtgagtccg gtggcatcga caaggaacct atgggcttca cttacaacgg tatccgtacc 420
aacggtgtga cttccgcctg ccgtaggtcc ggtagcagct tctacgctga gatgaagtgg 480
ctgctgagca acaccgacaa cgccgccttc cctcagatga ctaagtctta caagaacacc 540
cgtgagtccc ctgctatcgt cgtgtggggt atccaccaca gcgtgagcac tgctgaacag 600
actaagctct acggtagcgg taacaagctg gtgactgtgg gttctagcaa ctaccagcag 660
tccttcgttc cttcacctgg cgcccgccct caggtcaacg gtcagtccgg ccgcatcgac 720
ttccactggc tgatcctcaa cccaaacgac accgtcacct tctccttcaa cggcgccttc 780
atcgcccctg accgcgctag cttcctgcgt ggcaagagca tgggcatcca gtccggagtc 840
caggtggacg ccaactgtga aggtgactgt taccactccg gcggcaccat catcagcaac 900
ctgcctttcc agaacatcga cagccgtgct gtgggcaagt gtcctcgcta cgtgaagcag 960
cgctccctgc tgctggccac cggtatgaag aacgtgcctg aggtgcctaa ggcccgtggc 1020
ctgttcggcg ctatcgccgg tttcatcgaa aacggctggg agggtctgat cgacggctgg 1080
tacggtttcc gccaccaaaa cgcccagggt gaaggtaccg ctgctgacta caagagcaca 1140
cagtccgcca tcgaccaaat caccggcaag ctgaaccgcc tgatcgccaa gacaaaccag 1200
cagttcaagc tgatcgacaa cgaattcaac gaggtggaaa agcagatcgg taacgtgatc 1260
aactggacac gcgacagcat caccgaggtg tggtcctaca acgctgaact gctggtggct 1320
atggaaaacc agcacaccat cgacctcgct gacagcgaaa tggacaagct ctacgaacgc 1380
gttaagcgtc agctgcgcga aaacgccgaa gaagacggta caggttgttt cgaaatcttc 1440
cacaagtgtg acgacgactg catggcctcc atccgcaaca acacttacga ccaccgtaag 1500
taccgtgagg aagccatgca gaaccgtatc cagatcgacc ctgtgaagct gagcagcggt 1560
tacaaggacg tgatcctgtg gttcagcttc ggtgctagct gcttcatcct gctggccatc 1620
gtgatgggcc tggtgttcat ctgtgtgaag aacggtaaca tgaggtgtac catctgcatc 1680
taa 1683
<210> 8
<211> 1398
<212> DNA
<213> subtype H7 avian influenza virus
<400> 8
atgaacccta accagaagat cctgtgtact agcgctactg ctatcacaat cggcgctatc 60
accgtgctga tcggcatcgc taacatcggt ctgaacatcg gtctgcacct caagtccggc 120
tgtaactgta gccgtagcca gcctgagacc actaacacta gccagactat catcaacaac 180
tactacaacg agactaacat caccaacatc cagatgggag aacgcacctc ccgtaacttc 240
aacaacctga ccaagggcct gtgcactatc aactcctggc acatctacgg taaagacaac 300
gccgtgcgta tcggtgagtc ctctgacgtt ctggtgactc gtgagcctta cgtgtcctgc 360
gacccagacg agtgtaagtt ctacgccctg tcccagggca ctaccatccg tggcaagcac 420
agcaacggca ctatccacga ccgctcccag taccgcgccc tgatctcctg gccactgtcc 480
tcaccaccaa cagtctacaa cagccgcgtt gagtgcatcg gttggtcatc caccagctgc 540
cacgacggca agagccgcat gagcatctgt atctccggtc ctaacaacaa cgcctccgcc 600
gtgatctggt acaaccgtcg ccctgtcgct gaaatcaaca catgggctcg taacatcctc 660
cgtacacagg agagcgaatg tgtgtgccac aacggtgtgt gtcctgtcgt gttcaccgac 720
ggcccagcta ccggccccgc cgacacacgt atctactact tcaaggaagg caagatcctg 780
aagtgggaaa gcctgaccgg caccgctaag cacatcgagg agtgctcctg ctacggcaag 840
cgcaccggaa tcacctgcac ctgtcgcgac aactggcagg gctccaaccg tcccgtgatc 900
cagatcgacc ctgtggccat gacccacaca tcccagtaca tctgttcccc tgtgctcaca 960
gacagccccc gtcctaacga cccaaacatc ggcaagtgca acgaccccta ccctggtaac 1020
aacaacaacg gcgtgaaggg attctcctac ttggacggtg acaacacctg gctcggtcgc 1080
accatctcca ctgctagccg tagcggctac gaaatgctga aggtgcccaa cgccctgacc 1140
gacgaccgca gcaagcctat ccagggtcag accatcgttc tcaacgctga ctggtccggc 1200
tacagcggtt ccttcatgga ctactgggcc gagggtgact gctaccgcgc ttgtttctac 1260
gtggaactga tccgtggcaa gcccaaggag gacaaggtgt ggtggacctc caacagcatc 1320
gtgtccatgt gtagctccac cgaattcctg ggccagtgga actggcctga cggtgctaag 1380
atcgagtact tcctgtaa 1398
<210> 9
<211> 759
<212> DNA
<213> subtype H7 avian influenza virus
<400> 9
atgagcctgc tgaccgaagt ggaaacctac gtgctgagca tcatccctag cggtcctctg 60
aaggctgaaa tcgctcagcg tctggaagac gtgttcgctg gcaagaacgc tgacctggag 120
gctctgatgg aatggatcaa gacccgtcct atcctgagcc cactgactaa gggtatcctg 180
ggtttcgtgt tcaccctgac cgtgcctagc gagcgcggcc tgcagcgccg tcgtttcgtg 240
cagaacgctc tgaacggtaa cggtgaccct aacaacatgg acaaggccgt gaagctgtac 300
aagaagctga agcgtgagat gacattccac ggcgctaagg aagtggctct gagctactcc 360
accggtgctc tggcctcctg catgggcctg atctacaacc gtatgggtac cgtgaccgct 420
gaaggcgctc tgggtctggt gtgtgctaca tgtgagcaga tcgctgacgc ccagcaccgc 480
agccaccgcc agatggctac taccaccaac cctctgatcc gccacgagaa ccgcatggtg 540
ctggctagca ccactgctaa ggccatggaa cagatggccg gtagcagcga gcaggctgct 600
gaagccatgg aagtcgctag ccaggctcgt cagatggtgc aggctatgcg caccgtgggt 660
acccacccta actcctccac cggtctgaag gacgacctga tcgagaacct gcaggcttac 720
cagaaccgta tgggtgtgca gctgcagcgt ttcaagtaa 759
Claims (10)
1. An H5 subtype avian influenza virus-like particle antigen, wherein the H5 subtype avian influenza virus-like particle antigen is assembled by H5 subtype avian influenza virus HA, NA and M1 antigen proteins, and the H5 subtype avian influenza virus HA, NA and M1 antigen proteins are respectively coded by nucleotide sequences shown in Seq ID No.1, 2 and 3 or degenerate sequences thereof or nucleotide sequences shown in Seq ID No.4, 5 and 6 or degenerate sequences thereof.
2. An avian influenza virus-like particle vaccine, wherein the avian influenza virus-like particle vaccine comprises an immunizing amount of one or two avian influenza virus-like particle antigens of H5 subtype according to claim 1 and a pharmaceutically acceptable carrier.
3. The avian influenza virus-like particle vaccine as claimed in claim 2, wherein the antigen content of the avian influenza virus-like particles of subtype H5 is HA titer is more than or equal to 6log 2; preferably, the antigen content of the H5 subtype avian influenza virus-like particle is HA titer 6log 2-8 log 2.
4. The avian influenza vims-like particle vaccine of claim 2, wherein said pharmaceutically acceptable carrier comprises an adjuvant comprising: (1) mineral oil, alumina gel adjuvant, saponin, alfvudine, DDA; (2) water-in-oil emulsion, oil-in-water emulsion, water-in-oil-in-water emulsion; or (3) a copolymer of a polymer of acrylic acid or methacrylic acid, maleic anhydride and an alkenyl derivative; and one or more of RIBI adjuvant system, Block co-polymer, SAF-M, monophosphoryl lipid A, Avridine lipid-amine adjuvant, Escherichia coli heat-labile enterotoxin, cholera toxin, IMS 1314, muramyl dipeptide and Gel adjuvant; preferably, the saponin is Quil A, QS-21, GPI-0100; the concentration of the adjuvant ranges from 5% to 70% V/V, preferably from 30% to 70%, more preferably 66% V/V.
5. The avian influenza virus-like particle vaccine of claim 2, further comprising an immunizing amount of H7 subtype avian influenza virus-like particle antigen, wherein the H7 subtype avian influenza virus-like particle antigen is assembled from HA, NA and M1 antigen proteins of H7 subtype avian influenza virus.
6. The avian influenza virus-like particle vaccine of claim 5, wherein the H7 subtype avian influenza virus HA, NA and M1 antigen proteins are encoded by the nucleotide sequences shown in Seq ID No.7, 8 and 9 or degenerate sequences thereof, respectively.
7. The avian influenza virus-like particle vaccine of claim 5, wherein the antigen content of the avian influenza virus-like particles of subtype H7 is HA titer is more than or equal to 6log 2; preferably, the antigen content of the H7 subtype avian influenza virus-like particle is HA titer 6log 2-8 log 2.
8. A method of making the avian influenza virus-like particle vaccine of claim 2, wherein the method comprises:
cloning the HA, NA and M1 antigen protein genes of the avian influenza virus to the same vector;
transforming or transducing and recombining the vector obtained in the step (1) to obtain a recombinant baculovirus plasmid containing the HA, NA and M1 antigen protein genes;
step (3) transfecting the recombinant baculovirus plasmid containing the HA, NA and M1 antigen protein gene obtained in the step (2) into an insect cell sf9, and expressing the HA, NA and M1 antigen protein in series; and
and (4) separating the avian influenza virus-like particle antigen which is released to the supernatant of an extracellular culture medium after the self-assembly in insect cells is finished and assembled by the HA, NA and M1 antigen proteins, adding an adjuvant, and uniformly mixing to obtain the vaccine composition.
9. The method according to claim 8, wherein the vector in the step (1) is pFastBac I; the baculovirus plasmid in the step (2) is Bacmid.
10. The use of the avian influenza virus-like particle vaccine according to claims 2-6 in the preparation of a medicament for the prevention and/or treatment of a disease caused by an avian influenza virus; preferably, the avian influenza virus is an H5 subtype and/or an H7 subtype avian influenza virus.
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